PROJECT SUMMARY/ABSTRACT: The Acute Respiratory Distress Syndrome (ARDS) is significantly heterogeneous in both severity and response to treatment. While some treatments (PEEP, prone position) are more beneficial in severe cases, severity alone is insufficient to fully identify patient subgroups. Statistical analysis of clinical trial data has identified hyper-inflammatory and non-inflammatory endotypes with the former being associated with worse outcomes. However, such statistical approaches do not provide direct insight into mechanism. Here we propose to use a novel in vitro force microscopy platform to establish a mechanistic link between hyper inflammatory ARDS, endothelial disruption, and intercellular mechanical stress. We hypothesize that constellations of biomarkers impact the clinical phenotype by altering the mechanical state of the endothelium. Moreover, our preliminary data suggests that endothelial disruption results not from a disruption of a localized balance of forces at cell-cell junctions, but rather from a global reorganization of stress transmission within the endothelial layer. This mechanical reorganization moves the endothelium from an elastic phase, characterized by short- range forces and tolerant of large deformations prior to yielding, to a rigid phase, characterized by longer ranged forces and vulnerable to fracture. We further hypothesize that (i) endothelial mechanical states differ in their response to exogenous forces such as stretch or shear flow (ii) that the endothelial mechanical state may be defined in vitro by the set of cell-generated forces and the endothelial permeability and (iii) specific endothelial mechanical endotypes may be mapped to specific constellations of inflammatory biomarkers. In sum, we hypothesize that endothelial mechanical state may provide a causal link between measurable biomarkers and patient outcomes. The non-inflammatory endotype is characterized by an endothelium tolerant of physiologic levels of stretch and hyper inflammatory ARDS is characterized by an endothelium in which even low magnitude exogenous forces result in increased permeability.